J. Nat. Prod. 2005, 68, 935-937
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Taxezopidines M and N, Taxoids from the Japanese Yew, Taxus cuspidata Hiroshi Morita, Izumi Machida, Yusuke Hirasawa, and Jun’ichi Kobayashi* Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received March 15, 2005
Two new taxoids, taxezopidines M (1) and N (2), have been isolated from seeds of the Japanese yew, Taxus cuspidata, and new structures were elucidated on the basis of spectroscopic data. The absolute configuration of a 3-N,N-(dimethylamino)-3-phenylpropanoyl group in 1 and 2 was determined to be R in each case by chiral HPLC analysis. The effect of 1 and 2 on the CaCl2-induced depolymerization of microtubules was investigated. Since the discovery of anticancer activity of Taxol (paclitaxel), much attention has been paid to the isolation of new taxane diterpenoids from various species of yews.1,2 In our continuing search for bioactive taxoids, we have isolated previously a series of new taxoids, taxuspines A-H and J-Z3 and taxezopidines A-H and J-L,4-6 from the stems, leaves, and seeds of the Japanese yew, Taxus cuspidata Sieb. et Zucc. (Taxaceae). Further investigation on an extract of the seeds of this yew has led to the isolation of two new taxoids, taxezopidines M (1) and N (2). In this paper, the isolation, structure elucidation, and biological evaluation of 1 and 2 are described. The methanolic extract of seeds of T. cuspidata collected at Sapporo was partitioned between EtOAc and 3% tartaric acid. Water-soluble materials, which were adjusted to pH 10 with saturated Na2CO3, were extracted with CHCl3. The CHCl3-soluble materials were subjected to silica gel column chromatography and then C18 HPLC to afford taxezopidines M (1, 0.0001%) and N (2, 0.0001%) together with the known taxoids 2-O-deacetyl taxine II (0.0001%)7 and 1-deoxytaxine B (0.0014%).8
Taxezopidine M (1) was obtained as a colorless solid, and the molecular formula was established to be C37H49NO10 by HRFABMS [m/z 668.3437 (M + H)+, ∆ +0.2 mmu]. IR absorptions implied that 1 possesses hydroxy (3440 cm-1) and ester (1730 cm-1) groups. Analysis of the 1H and 13C NMR data and the HMQC spectrum provided evidence that 1 possesses three acetyl groups, one tetrasubstituted olefin, * To whom correspondence should be addressed. Tel: (011)706-4985. Fax: (011)706-4989. E-mail:
[email protected].
10.1021/np0500880 CCC: $30.25
one exomethylene, one hemiacetal carbon, four oxymethines, one oxymethylene, five methyl groups, four methylenes, three methines, one ester carbonyl, and six aromatic carbons. The 1H and 13C NMR (Table 1) signals (δH 2.82, 3.28, 3.64, 4.83, and 7.53; δC 37.0, 41.8, 67.8, 132.1, and 170.5) of 1 indicated the presence of a 3-N,N-(dimethylamino)-3phenylpropanoyl group (Winterstein’s acid)9 at C-5, which was implied by a HMBC correlation for H-5 to C-21. Connectivities of C-1 to C-3, C-5 to C-7, C-9 to C-10, C-14 to C-1, and C-22 to C-23 were deduced from the 1H-1H COSY and HOHAHA (Figure 1) spectra. HMBC correlations (Figure 1) of H3-18 to C-11, C-12, and C-13 and from H3-16 to C-1, C-11, and C-15 revealed that Me-18 and Me-16 are attached to C-12 and C-15, respectively. Three acetoxy groups were placed at C-2, C-9, and C-10 on the basis of HMBC correlations, while a hydroxy group was attached to C-13 from comparison of the 13C NMR chemical shift of C-13 (δ 97.2) with those of hemiacetal carbons (δ 96-98) reported previously.10 Connectivities among C-3, C-7, C-9, and Me-19 through C-8 were provided by HMBC correlations for H3-19 to C-3, C-7, C-9, and C-8. The HMBC correlation of H-10 to C-15 revealed the connection between C-10 and C-11. The exomethylene was inserted between C-3 and C-5 by HMBC correlations of H2-20 to C-3 and C-5. Thus, the gross structure of taxezopidine M was elucidated to be 1. The relative stereochemistry of 1 was deduced from NOESY data (Figure 2). A boat-chair conformation of the eight-membered ring (C-1-C-3, C-8-C-11, and C-15) was elucidated from the coupling constant (10.7 Hz) between H-9 and H-10 and NOESY correlations. The NOESY correlation between H-1 and H-17a indicated that both protons were quasi-axial and the oxymethylene (C-17) at C-15 was β-oriented, while the correlations of H-3 to H-14a and H3-18 revealed that 1 has a cage-like backbone conformation. Taxezopidine N (2) was shown to have the molecular formula C39H51NO13 by HRFABMS [m/z 742.3442 (M + H)+, ∆ -0.4 mmu]. The IR spectrum indicated the presence of hydroxy (3360 cm-1) and ester (1740 cm-1) groups. The 1H and 13C NMR (Table 1) spectroscopic data of 2 resembled those of taxezopidine L.6 An HMBC correlation (Figure 3) between H-17a and C-12 and the proton signals (δH 4.12 and 3.82, d, J ) 8.1 Hz; H-17a and H-17b) at C-17 revealed the presence of a tetrahydrofuran ring (C-11, C-12, C-15, C-17, and O-1) fused to a six-membered ring (C-1 and C-11-C-15). The 1H and 13C NMR signals of C-21-C-29 (δ 2.88, 3.02, 3.64, H 3.72, 5.00, and 7.61; δC 35.9, 40.8, 67.5, 130.6, 130.9, 132.9,
© 2005 American Chemical Society and American Society of Pharmacognosy Published on Web 05/14/2005
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Journal of Natural Products, 2005, Vol. 68, No. 6
Table 1. 1H and (2) in CD3ODa
13C
NMR Data of Taxezopidines M (1) and N 1
1H
position 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17a 17b 18 19 20a 20b 21 22a 22b 23 OAc-2
2.14 (1H, m) 5.53 (1H, d, 4.9) 2.96 (1H, d, 4.9) 5.21 (1H, brs) 1.76 (1H, m) 1.76 (1H, m) 1.76 (2H, m) 5.75 (1H, d, 10.7) 6.07 (1H, d, 10.7)
2.14 (1H, m) 1.48 (1H, d, 15.4) 1.49 (3H, s) 3.52 (1H, d, 8.1) 3.08 (1H, d, 8.1) 2.27 (3H, s) 0.89 (3H, s) 5.32 (1H, s) 5.12 (1H, s) 3.64 (1H, m) 3.28 (1H, m) 4.83 (1H, m) 2.04 (3H, s)
2 13C
49.2 71.3 43.7 142.6 81.1 28.9 28.6 44.8 77.5 71.2 130.4 143.4 97.2 35.8 38.6 17.6 75.2 12.5 17.7 119.9 170.5 37.0 67.8 21.3 171.2
OAc-7
1H
2.54 (1H, m) 5.59 (1H, m) 3.29 (1H, m) 5.17 (1H, m) 1.93 (1H, m) 1.55 (1H, m) 5.29 (1H, m) 5.27 (1H, d, 2.6) 5.35 (1H, d, 2.6)
3.23 (1H, m) 2.51 (1H, m) 1.53 (3H, s) 4.12 (1H, d, 8.1) 3.82 (1H, d, 8.1) 1.12 (3H, s) 1.05 (3H, s) 5.37 (1H, s) 4.63 (1H, s) 3.72 (1H, m) 3.64 (1H, m) 5.00 (1H, m) 1.99 (3H, s) 2.17 (3H, s)
OAc-9
2.07 (3H, s)
OAc-10
2.00 (3H, s)
Ph NMe2-23 a
Notes
2.12 (3H, s)
7.53 (5H, m)
20.8 172.0 20.6 171.4 132.1
2.82 (6H, m)
41.8
3.02 (3H, m) 2.88 (3H, m)
2.02 (3H, s) 7.61 (5H, m)
13C
50.6 69.9 41.5 140.8 77.9 37.1 69.5 47.7 75.4 65.4 82.2 93.2 209.3 36.6
Figure 2. Selected NOESY correlations and relative stereochemistry for taxezopidine M (1).
50.4 16.0 82.7 12.3 13.2 117.0 169.0 35.9 67.5 21.2 170.7 21.4 171.6 20.9 172.4 21.2 171.1 130.6 130.9 132.9 40.8 40.8
δ in ppm.
Figure 1. Selected two-dimensional NMR correlations for taxezopidine M (1).
and 169.0) indicated the presence of a 3-N,N-(dimethylamino)-3-phenylpropanoyl group (Winterstein’s acid)9 at C-5, which was implied by the HMBC correlation from H-5 to C-21. Four acetoxy groups were attached to C-2, C-7, C-9, and C-10 based on the HMBC correlations and oxymethine proton resonances (δH 5.59, H-2; 5.29, H-7; 5.27, H-9; and 5.35, H-10). The presence of a ketone (δC 209.3) at C-13 was deduced from HMBC correlations of H3-18 and H-1 to C-13. HMBC correlations of H-20b to C-3 and C-5 indicated the presence of an exomethylene at C-4. Thus, the gross structure of taxezopidine N was assigned as 2.
Figure 3. Selected two-dimensional NMR correlations for taxezopidine N (2).
Figure 4. Selected NOESY correlations and relative stereochemistry for taxezopidine N (2).
The relative stereochemistry of 2 was elucidated by NOESY data (Figure 4) and 1H-1H coupling constants. The absolute stereochemistry of a 3-N,N-(dimethylamino)-3phenylpropanoyl group (Winterstein’s acid) in taxezopidines M (1) and N (2) was determined to be R in both cases by chiral HPLC analysis (Sumichiral OA-5000) of the acid hydrolysates of 1 and 2. Microtubules polymerized in the presence of paclitaxel are resistant to depolymerization by Ca2+ ions.11 The effect of taxezopidines M (1) and N (2) was examined against the CaCl2-induced depolymerization of microtubules. Microtubule proteins were polymerized under normal polymerization conditions12 in the absence and the presence of paclitaxel, and after a 10 min incubation, CaCl2 was added. Microtubule polymerization and depolymerization were monitored by the increase and decrease in turbidity. The results are summarized in Figure 5 as the changes in the relative absorbance at 400 nm. The CaCl2-induced depolymerization of microtubules was inhibited by 5 µM paclitaxel. Taxezopidine M (1) reduced the depolymerization process at higher concentration (50 µM), while taxezopidine N (2) at the same concentration showed little effect. Taxezopidines M (1) and N (2) did not show cytotoxicity against murine lymphoma L1210 and human epidermoid carcinoma KB cells at 10 µg/mL.
Notes
Figure 5. Effect of taxezopidines M (1) and N (2) on Ca2+-induced microtubule depolymerization. Temperature was held at 37 °C, and changes in turbidity were monitored at 400 nm, with 4 mM CaCl2 added at 10 min.
Experimental Section General Experimental Procedures. Optical rotations were measured on a JASCO P-1030 polarimeter. UV spectra were recorded on a Shimadzu UV1600PC spectrophotometer and IR spectra on a JASCO FTIR-230 spectrometer. 1H and 2D NMR spectra were recorded on a 600 MHz spectrometer at 300 K, while 13C NMR spectra were measured on a 150 MHz spectrometer. Compounds 1 and 2 were prepared by dissolving 1.0 mg in 30 µL of CD3OD in 2.5 mm micro cells (Shigemi Co. Ltd., Tokyo, Japan), and chemical shifts were reported using residual CH3OH (δH 3.31 and δC 49.0) as internal standard. Standard pulse sequences were employed for the 2D NMR experiments. 1H-1H COSY, HOHAHA, and NOESY NMR spectra were measured with spectral widths of both dimensions of 4800 Hz, and 32 scans with two dummy scans were accumulated into 1K data points for each of 256 t1 increments. NOESY and HOHAHA spectra in the phase-sensitive mode were measured with a mixing time of 800 and 30 ms, respectively. For HMQC spectra in the phase-sensitive mode and HMBC spectra, a total of 256 increments of 1K data points were collected. For HMBC spectra with the Z-axis PFG, a 50 ms delay time was used for long-range C-H coupling. Zerofilling to 1 K for F1 and multiplication with squared cosinebell windows shifted in both dimensions were performed prior to 2D Fourier transformation. FABMS was measured on a JEOL HX-110 spectrometer by using a glycerol matrix. Plant Material. The Japanese yew T. cuspidata Sieb. et Zucc. was collected at Sapporo, Hokkaido, in 2003. The botanical identification was made by Mr. N. Yoshida, Health Sciences University of Hokkaido. A voucher specimen (No. 030001) has been deposited in the herbarium of Hokkaido University. Extraction and Isolation. The MeOH extract (1373 g) of the seeds (10 kg) of the yew was partitioned between EtOAc and 3% tartaric acid. Water-soluble materials, after being adjusted at pH 10 with saturated Na2CO3, were partitioned with CHCl3. The CHCl3-soluble materials (3.8 g) were subjected to passage over a silica gel column (MeOH-CHCl3EtOAc, 100:1:0.5 f MeOH f MeOH-TFA, 100:1) and then C18 HPLC (YMC-Pack ODS AM-323, 5 µm, 20 × 250 mm; flow rate 8.0 mL/min; UV detection at 254 nm; eluent, 30-40% CH3CN-0.1% TFA) to afford taxezopidines M (1, 1.2 mg) and N (2, 2.2 mg), 2-O-deacetyltaxine II (1.2 mg), and 1-deoxytaxine B (14.3 mg).
Journal of Natural Products, 2005, Vol. 68, No. 6 937
Taxezopidine M (1): colorless solid; [R]23D +30° (c 0.4, MeOH); UV (MeOH) λmax (log ) 276 (3.0), 204 nm (4.0); IR (film) νmax 3440, 1730, 1240 cm-1; 1H and 13C NMR (Table 1); FABMS m/z 668 (M + H)+; HRFABMS m/z 668.3437 (M + H)+, calcd for C37H50NO10 668.3435. Taxezopidine N (2): colorless solid; [R]23D +16° (c 1.0, MeOH); UV (MeOH) λmax (log ) 276 (3.0), 205 nm (3.9); IR (film) νmax 3360, 1740, 1230 cm-1; 1H and 13C NMR (Table 1); FABMS m/z 742 (M + H)+; HRFABMS m/z 742.3442 (M + H)+, calcd for C39H52NO13 742.3446. Determination of Absolute Configuration of 3-N,NDimethylamino-3-phenylpropionic Acid in Taxezopidines M (1) and N (2). Taxezopidine M (1; 0.2 mg) was refluxed in 10% aqueous HCl (0.1 mL) for 1 h. The reaction mixture was extracted with Et2O (1 mL × 2), and the aqueous layer was concentrated under reduced pressure. The residue was dissolved in H2O for chiral HPLC analysis. The chiral HPLC analysis was carried out using a Sumichiral OA-5000 column (Sumitomo Chemical Industry, 0.46 × 15 cm; flow rate 1.0 mL/min; UV detection at 254 nm; eluent 2.0 mM CuSO4 in H2O-CH3OH, 97:3). The retention times of standard 3-(S)- and 3-(R)-N,N-(dimethylamino)-3-phenylpropanoic acids13 were 12.8 and 14.7 min, respectively, and that of 3-N,N-(dimethylamino)3-phenylpropanoic acid contained in the hydrolysate of 1 was found to be 14.7 min. According to the same procedure as described above, the retention time of 3-N,N-(dimethylamino)3-phenylpropanoic acid contained in the hydrolysate of taxezopidine N (2) was found to be 14.7 min. Microtubule Assembly Assay.14 Microtubule assembly was monitored spectroscopically using a spectrophotometer equipped with a thermostatically regulated liquid circulator. The temperature was held at 37 °C, and changes in turbidity were monitored at 400 nm. The turbidity changes were monitored throughout the incubation time. Acknowledgment. The authors thank Ms. S. Oka and Ms. M. Kiuchi, Center for Instrumental Analysis, Hokkaido University, for measurements of FABMS, and Mr. N. Yoshida, Health Sciences University of Hokkaido, for the botanical identification. This work was supported partially by a Grantin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a grant from the Shorai Foundation for Science and Technology. References and Notes (1) (a) Shigemori, H.; Kobayashi, J. J. Nat. Prod. 2004, 67, 245-256. (b) Baloglu, E.; Kingston, D. G. I. J. Nat. Prod. 1999, 62, 1448-1472, and references therein. (2) Appendino, G. Nat. Prod. Rep. 1995, 349-360. (3) Kobayashi, J.; Shigemori, H. Heterocycles 1998, 47, 1111-1133, and references therein. (4) Wang, X.-x.; Shigemori, H.; Kobayashi, J. J. Nat. Prod. 1998, 61, 474479. (5) Wang, X.-x.; Shigemori, H.; Kobayashi, J. Tetrahedron Lett. 1998, 38, 7587-7588. (6) Shigemori, H.; Sakurai, C. A.; Hosoyama, H.; Kobayashi, A.; Kajiyama, S.; Kobayashi, J. Tetrahedron 1999, 55, 2553-2558. (7) Shi, Q.; Oritani, T.; Sugiyama, T.; Oritani, T. Nat. Prod. Lett. 2000, 14, 265-272. (8) Van Rozendaal, E. L. M.; Veldhuis, H.; Beusker, P. H.; Beek, T. A. Herba Polo. 1998, 44, 227-231. (9) Winterstein, E.; Guyer, A. Z. Physiol. Chem. 1923, 128, 175-229. (10) Sun, H.-D.; Lin, Z.-W.; Niu, F.-D.; Lin, L.-Z.; Chai, H.; Pezzuto, J. M.; Cordell, G. A. J. Nat. Prod. 1994, 57, 1424-1429. (11) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665-667. (12) Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 765-768. (13) Graf, E.; Boeddeker, H. Ann. Chem. 1958, 613, 111-120. (14) Kobayashi, J.; Suzuki, H.; Shimbo, K.; Takeya, K.; Morita, H. J. Org. Chem. 2001, 66, 6626-6633.
NP0500880
